This invention relates to enzyme inhibitors, and more particularly to histone deacetylase inhibitors.
DNA in the nucleus of the cell exists as a hierarchy of compacted chromatin structures. The basic repeating unit in chromatin is the nucleosome. The nucleosome consists of a histone octomer of proteins in the nucleus of the cell around which DNA is twice wrapped. The orderly packaging of DNA in the nucleus plays an important role in the functional aspects of gene regulation. Covalent modifications of the histones have a key role in altering chromatin higher order structure and function and ultimately gene expression. The covalent modification of histones occurs by enzymatically mediated processes, such as acetylation.
Regulation of gene expression through the inhibition of the nuclear enzyme histone deacetylase (HDAC) is one of several possible regulatory mechanisms whereby chromatin activity can be affected. The dynamic homeostasis of the nuclear acetylation of histones can be regulated by the opposing activity of the enzymes histone acetyl transferase (HAT) and histone deacetylase (HDAC). Transcriptionally silent chromatin can be characterized by nucleosomes with low levels of acetylated histones. Acetylation of histones reduces its positive charge, thereby expanding the structure of the nucleosome and facilitating the interaction of transcription factors to the DNA. Removal the acetyl group restores the positive charge condensing the structure of the nucleosome. Acetylation of histone-DNA activates transcription of DNA""s message, an enhancement of gene expression. Histone deacetylase can reverse the process and can serve to repress gene expression. See, for example, Grunstein, Nature 389, 349-352 (1997); Pazin et al., Cell 89, 325-328 (1997); Wade et al., Trends Biochem. Sci. 22, 128-132 (1997); and Wolffe, Science 272, 371-372 (1996).
Histone deacetylase is a metallo-enzyme with zinc at the active site. Compounds having a zinc-binding moiety, such as, for example, a hydroxamic acid group, can inhibit histone deacetylase. Histone deacetylase inhibition can repress gene expression, including expression of genes related to tumor suppression. Accordingly, inhibition of histone deacetylase can provide an alternate route for treating cancer, hematological disorders, e.g., hemoglobinopathies, and genetic related metabolic disorders, e.g., cystic fibrosis and adrenoleukodystrophy.
In one aspect, hydroxamic acid-containing compounds have a structure of formula (I): 
A is a cyclic moiety selected from the group consisting of C3-14 cycloalkyl, 3-14 membered heterocycloalkyl, C4-14 cycloalkenyl, 3-14 membered heterocycloalkenyl (e.g., C3-8 cycloalkyl, 3-8 membered heterocycloalkyl, C4-8 cycloalkenyl, 3-8 membered heterocycloalkenyl), monocyclic aryl, or monocyclic heteroaryl. Each of these cyclic moieties is optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl, alkylcarbonylamino, aminocarbonyl, alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl. Each of X1 and X2, independently, is O or S. Y1 is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N(Ra)xe2x80x94, xe2x80x94N(Ra)xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94N(Ra)xe2x80x94, xe2x80x94N(Ra)xe2x80x94C(O)xe2x80x94N(Rb)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, or a bond wherein each of Ra and Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. Y2 is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N(Rc)xe2x80x94, xe2x80x94N(Rc)xe2x80x94C(O)xe2x80x94Oxe2x80x94C(O)xe2x80x94N(Rc)xe2x80x94, xe2x80x94N(Rc)xe2x80x94C(O)xe2x80x94N(Rd)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, or xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94 wherein each of Rc and Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. L is (1) a saturated straight C1-12 hydrocarbon chain substituted with C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy, halo, carboxyl, amino, nitro, cyano, C3-6 cycloalkyl, 3-6 membered heterocycloalkyl, monocyclic aryl, 5-6 membered heteroaryl, C1-4 alkylcarbonyloxy, C1-4 alkyloxycarbonyl, C1-4 alkylcarbonyl, formyl, C1-4 alkylcarbonylamino, or C1-4 aminocarbonyl, or at least two hydroxyl; and further optionally interrupted by xe2x80x94Oxe2x80x94, xe2x80x94N(Re)xe2x80x94, xe2x80x94N(Re)xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94N(Re)xe2x80x94, xe2x80x94N(Re)xe2x80x94C(O)xe2x80x94N(Rf)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, or xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94 wherein each of Re and Rf, independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl; or L is (2) an unsaturated straight C4-12 hydrocarbon chain containing at least two double bonds, at least one triple bond, or at least one double bond and one triple bond, where the unsaturated hydrocarbon chain is optionally substituted with C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy, hydroxyl, halo, carboxyl, amino, nitro, cyano, C3-6 cycloalkyl, 3-6 membered heterocycloalkyl, monocyclic aryl, 5-6 membered heteroaryl, C1-4 alkylcarbonyloxy, C1-4 alkyloxycarbonyl, C1-4 alkylcarbonyl, formyl, C1-4 alkylcarbonylamino, or C1-4 aminocarbonyl; and further being optionally interrupted by xe2x80x94Oxe2x80x94, xe2x80x94N(Rg)xe2x80x94, xe2x80x94N(Rg)xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94N(Rg)xe2x80x94, xe2x80x94N(Rg)xe2x80x94C(O)xe2x80x94N(Rh)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, or xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94 wherein each of Rg and Rh, independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. R1 is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, haloalkyl, or an amino protecting group; and R2 is hydrogen, alkyl, hydroxylalkyl, haloalkyl, or a hydroxyl protecting group.
In another aspect, hydroxamic acid-containing compounds have a structure of formula (I), supra. A is a cyclic moiety selected from the group consisting of monocyclic aryl or monocyclic heteroaryl. Each of the cyclic moieties is optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, or amino. Each of X1 and X2, independently, is O or S. Y1 is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N(Ra)xe2x80x94, xe2x80x94N(Ra)xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94N(Ra)xe2x80x94, xe2x80x94N(Ra)xe2x80x94C(O)xe2x80x94N(Rb)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, or a bond, where each of Ra and Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. Y2 is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N(Rc)xe2x80x94, xe2x80x94N(Rc)xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94N(Rc)xe2x80x94, xe2x80x94N(Rc)xe2x80x94C(O)xe2x80x94N(Rd)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, or xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94; each of Rc and Rd, independently, being hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. L is (1) a saturated straight C3-10 hydrocarbon chain substituted with C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy, or amino, and further optionally interrupted by xe2x80x94Oxe2x80x94 or xe2x80x94N(Re)xe2x80x94, where Re is hydrogen, alkyl, hydroxylalkyl, or haloalkyl; or L is (2) an unsaturated straight C4-10 hydrocarbon chain containing at least two double bonds, at least one triple bond, or at least one double bond and one triple bond; said unsaturated hydrocarbon chain being optionally substituted with C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy, or amino, and further optionally interrupted by xe2x80x94Oxe2x80x94 or xe2x80x94N(Rf)xe2x80x94, where Rf is hydrogen, alkyl, hydroxylalkyl, or haloalkyl. Each of R1 and R2, independently, is hydrogen, alkyl, hydroxylalkyl, or haloalkyl.
In certain embodiments, R1 is hydrogen, R2 is hydrogen, X1 is O, X2 is O, or Y1 is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94N(Raxe2x80x94, or a bond, and Y2 is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, or xe2x80x94N(Rc)xe2x80x94. L can be a saturated straight C4-10 hydrocarbon chain, or C5-8 hydrocarbon chain (e.g., a saturated straight C5 hydrocarbon chain, a saturated straight C6 hydrocarbon chain, or a saturated straight C7 hydrocarbon chain), substituted with C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy, or amino, and further optionally interrupted by xe2x80x94Oxe2x80x94 or xe2x80x94N(Rc)xe2x80x94. In other embodiments, L is an unsaturated straight C4-10 hydrocarbon chain, or an unsaturated straight C4-8 hydrocarbon chain, containing 2-5 double bonds, or 1-2 double bonds and 1-2 triple bonds, optionally substituted with C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, or C1-4 alkoxy, and further being optionally interrupted by xe2x80x94Oxe2x80x94 or xe2x80x94N(Rg)xe2x80x94. In certain embodiments, L can be xe2x80x94(CHxe2x95x90CH)mxe2x80x94 where m is 2 or 3 or L can be xe2x80x94Cxe2x89xa1Cxe2x80x94(CHxe2x95x90CH)nxe2x80x94 where n is 1 or 2. A can be phenyl, furyl, thienyl, pyrrolyl, or pyridyl or A can be phenyl optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, or amino.
In a further aspect, hydroxamic acid-containing compounds have a structure of formula (II): 
A is a cyclic moiety selected from the group consisting of monocyclic aryl or monocyclic heteroaryl. Each of the cyclic moieties is optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, or amino. Each of X1 and X2, independently, is O or S. Each of R1 and R2, independently, is hydrogen, alkyl, hydroxylalkyl, or haloalkyl. Each of R3, R4, R5, R6, R7, R8, R9 and R10, independently, is hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy, hydroxyl, halo, hydroxylC1-4 alkyl, haloC1-4 alkyl, or amino, and each of a, b, c, d, e, and f, independently, is 0 or 1. Note that at least one of b, c, d, and e cannot be zero. In certain embodiments, a is 0, f is 0, or the total number of b, c, d, and e is 3 or 4. In other embodiments, each of R3, R4, R5, R6, R7, R8, R9 and R10, independently, is hydrogen, C1-4 alkyl, C1-4 alkoxy, hydroxyl, hydroxylC1-4 alkyl, or amino. Each of R5, R6, R7, and R8, independently can be hydrogen, C1-4 alkyl, C1-4 alkoxy, hydroxyl, hydroxylC1-4 alkyl, or amino, Each of R3, R4, R9 and R10, independently, can be hydrogen.
In another aspect hydroxamic acid-containing compounds have the structure of formula (I), supra. A is a saturated branched C3-14 hydrocarbon chain or an unsaturated branched C3-14 hydrocarbon chain optionally interrupted by xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N(Ra)xe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94N(Ra)xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94N(Ra)xe2x80x94, xe2x80x94N(Ra)xe2x80x94SO2xe2x80x94, xe2x80x94SO2xe2x80x94N(Ra)xe2x80x94, xe2x80x94N(Ra)xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94N(Ra)xe2x80x94, xe2x80x94N(Ra)xe2x80x94C(O)xe2x80x94N(Rb)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, or xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, where each of Ra and Rb, independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. Each of the saturated and the unsaturated branched hydrocarbon chain is optionally substituted with alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl, alkylcarbonylamino, aminocarbonyl, alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl. Each of X1 and X2, independently, is O or S. Each of Y1 and Y2, independently, is xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94N(Rc)xe2x80x94, xe2x80x94N(Rc)xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94N(Rc)xe2x80x94, xe2x80x94N(Rc)xe2x80x94C(O)xe2x80x94N(Rd)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, or a bond, where each of Rc and Rd, independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. L is a saturated straight C3-12 hydrocarbon or an unsaturated straight C4-12 hydrocarbon chain, said hydrocarbon chain being optionally substituted with C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy, hydroxyl, halo, carboxyl, amino, nitro, cyano, C3-6 cycloalkyl, 3-6 membered heterocycloalkyl, monocyclic aryl, 5-6 membered heteroaryl, C1-4 alkylcarbonyloxy, C1-4 alkyloxycarbonyl, C1-4 alkylcarbonyl, formyl, C1-4 alkylcarbonylamino, or C1-4 aminocarbonyl; and further optionally interrupted by xe2x80x94Oxe2x80x94, xe2x80x94N(Re)xe2x80x94, xe2x80x94N(Re)xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94N(Re)xe2x80x94, xe2x80x94N(Re)xe2x80x94C(O)xe2x80x94N(Rf)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, or xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, where each of Re and Rf, independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. R1 is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl, haloalkyl, or an amino protecting group; and R2 is hydrogen, alkyl, hydroxylalkyl, haloalkyl, or a hydroxyl protecting group.
Set forth below are some examples of a hydroxamic acid-containing compound of the present invention: benzylthioglycoloylhydroxamic acid, N-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid, 3-methyl-5-phenyl-2,4-pentadienoyl hydroxamic acid, 4-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid, 4-chloro-5-phenyl-2,4-pentadienoylhydroxamic acid, 5-(4-dimethylaminophenyl)-2,4-pentadienoylhydroxamic acid, 5-phenyl-2-en-4-yn-pentanoylhydroxamic acid, 5-(2-furyl)-2,4-pentadienoylhydroxamic acid, N-methyl-6-phenyl-3,5-hexadienoylhydroxamic acid, and 7-phenyl-2,4,6-hepta-trienoylhydroxamic acid.
A salt of any of the compounds of the invention can be prepared. For example, a pharmaceutically acceptable salt can be formed when an amino-containing compound of this invention reacts with an inorganic or organic acid. Some examples of such an acid include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. Examples of pharmaceutically acceptable salts thus formed include sulfate, pyrosulfate bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, and maleate. A compound of this invention may also form a pharmaceutically acceptable salt when a compound of this invention having an acid moiety reacts with an inorganic or organic base. Such salts include those derived from inorganic or organic bases, e.g., alkali metal salts such as sodium, potassium, or lithium salts; alkaline earth metal salts such as calcium or magnesium salts; or ammonium salts or salts of organic bases such as morpholine, piperidine, pyridine, dimethylamine, or diethylamine salts.
It should be recognized that a compound of the invention can contain chiral carbon atoms. In other words, it may have optical isomers or diastereoisomers.
Alkyl is a straight or branched hydrocarbon chain containing 1 to 10 (preferably, 1 to 6; more preferably 1 to 4) carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylhexyl, and 3-ethyloctyl.
The terms xe2x80x9calkenylxe2x80x9d and xe2x80x9calkynylxe2x80x9d refer to a straight or branched hydrocarbon chain containing 2 to 10 carbon atoms and one or more (preferably, 1-4 or more preferably 1-2) double or triple bonds, respectively. Some examples of alkenyl and alkynyl are allyl, 2-butenyl, 2-pentenyl, 2-hexenyl, 2-butynyl, 2-pentynyl, and 2-hexynyl.
Cycloalkyl is a monocyclic, bicyclic or tricyclic alkyl group containing 3 to 14 carbon atoms. Some examples of cycloalkyl are cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl. Heterocycloalkyl is a cycloalkyl group containing at least one heteroatom (e.g., 1-3) such as nitrogen, oxygen, or sulfur. The nitrogen or sulfur may optionally be oxidized and the nitrogen may optionally be quaternized. Examples of beterocycloalkyl include piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrofuryl, and morpholinyl. Cycloalkenyl is a cycloalkyl group containing at least one (e.g., 1-3) double bond. Examples of such a group include cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl, and cyclooctenyl groups. By the same token, heterocycloalkenyl is a cycloalkenyl group containing at least one heteroatom selected from the group of oxygen, nitrogen or sulfur.
Aryl is an aromatic group containing a 5-14 ring and can contain fused rings, which may be saturated, unsaturated, or aromatic. Examples of an aryl group include phenyl, naphthyl, biphenyl, phenanthryl, and anthracyl. If the aryl is specified as xe2x80x9cmonocyclic aryl,xe2x80x9d if refers to an aromatic group containing only a single ring, i.e., not a fused ring.
Heteroaryl is aryl containing at least one (e.g., 1-3) heteroatom such as nitrogen, oxygen, or sulfur and can contain fused rings. Some examples of heteroaryl are pyridyl, furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, and benzthiazolyl.
The cyclic moiety can be a fused ring formed from two or more of the just-mentioned groups. Examples of a cyclic moiety having fused rings include fluorenyl, dihydrodibenzoazepine, dibenzocycloheptenyl, 7H-pyrazino[2,3-c]carbazole, or 9,10-dihydro-9,10-[2]buteno-anthracene.
Amino protecting groups and hydroxy protecting groups are well-known to those in the art. In general, the species of protecting group is not critical, provided that it is stable to the conditions of any subsequent reaction(s) on other positions of the compound and can be removed without adversely affecting the remainder of the molecule. In addition, a protecting group may be substituted for another after substantive synthetic transformations are complete. Examples of an amino protecting group include, but not limited to, carbamates such as 2,2,2-trichloroethylcarbamate or tertbutylcarbamate. Examples of a hydroxyl protecting group include, but not limited to, ethers such as methyl, t-butyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl, trityl, methoxymethyl, 2-methoxypropyl, methoxyethoxymethyl, ethoxyethyl, tetrahydropyranyl, tetrahydrothiopyranyl, and trialkylsilyl ethers such as trimethylsilyl ether, triethylsilyl ether, dimethylarylsilyl ether, triisopropylsilyl ether and t-butyldimethylsilyl ether; esters such as benzoyl, acetyl, phenylacetyl, formyl, mono-, di-, and trihaloacetyl such as chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl; and carbonates including but not limited to alkyl carbonates having from one to six carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl; isobutyl, and n-pentyl; alkyl carbonates having from one to six carbon atoms and substituted with one or more halogen atoms such as 2,2,2-trichloroethoxymethyl and 2,2,2-trichloro-ethyl; alkenyl carbonates having from two to six carbon atoms such as vinyl and allyl; cycloalkyl carbonates having from three to six carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and phenyl or benzyl carbonates optionally substituted on the ring with one or more C1-6 alkoxy, or nitro. Other protecting groups and reaction conditions can be found in T. W. Greene, Protective Groups in Organic Synthesis, (3rd, 1999, John Wiley and Sons, New York, N.Y.).
Note that an amino group can be unsubstituted (i.e., xe2x80x94NH2), mono-substituted (i.e., xe2x80x94NHR), or di-substituted (i.e., xe2x80x94NR2). It can be substituted with groups (R) such as alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. Halo refers to fluoro, chloro, bromo, or iodo.
Inhibition of a histone deacetylase in a cell is determined by measuring the level of acetylated histones in the treated cells and measuring the level of acetylated histones in untreated cells and comparing the levels. If the level of histone acetylation in the treated cells increases relative to the untreated cells, histone deacetylase has been inhibited.
Some disorders or physiological conditions may be mediated by hyperactive histone deacetylase activity. A disorder or physiological condition that is mediated by histone deacetylase refers to a disorder or condition wherein histone deacetylase plays a role in triggering the onset thereof. Examples of such disorders or conditions include, but not limited to, cancer, hemoglobinopathies (e.g., thalassemia or sickle cell anemia), cystic fibrosis, protozoan infection, adrenoleukodystrophy, alpha-1 anti-trypsin, retrovirus gene vector reactivation, wound healing, hair growth, peroxisome biogenesis disorder, and adrenoleukodystrophy.
Other features or advantages will be apparent from the following detailed description of several embodiments, and also from the appended claims.
A carboxylic acid-containing compound of the present invention can be prepared by any known methods in the art. For example, a compound of the invention having an unsaturated hydrocarbon chain between A and xe2x80x94C(xe2x95x90X1)xe2x80x94 can be prepared according to the following scheme: 
where Lxe2x80x2 is a saturated or unsaturated hydrocarbon linker between A and xe2x80x94CHxe2x95x90CHxe2x80x94 in a compound of the invention, and A and X1 has the same meaning as defined above. See Coutrot et al., Syn. Comm. 133-134 (1978). Briefly, butyllithium was added to an appropriate amount of anhydrous tetrahydrofuran (THF) at a very low temperature (e.g., xe2x88x9265xc2x0 C.). A second solution having diethylphosphonoacetic acid in anhydrous THF was added dropwise to the stirred butyllithium solution at the same low temperature. The resulting solution is stirred at the same temperature for an additional 30-45 minutes which is followed by the addition of a solution containing an aromatic acrylaldehyde in anhydrous THF over 1-2 hours. The reaction mixture is then warmed to room temperature and stirred overnight. It is then acidified (e.g., with HCl) which allows the organic phase to be separated. The organic phase is then dried, concentrated, and purified (e.g., by recrystallization) to form an unsaturated carboxylic acid-containing intermediate.
Alternatively, a carboxylic acid-containing compound can be prepared by reacting an acid ester of the formula Axe2x80x94Lxe2x80x2xe2x80x94C(xe2x95x90O)xe2x80x94O-lower alkyl with a Grignard reagent (e.g., methyl magnesium iodide) and a phosphorus oxychloride to form a corresponding aldehyde, which can be further oxidized (e.g., by reacting with silver nitrate and aqueous NaOH) to form an unsaturated carboxylic acid-containing intermediate.
Other types of carboxylic acid-containing compounds (e.g., those containing a linker with multiple double bonds or triple bonds) can be prepared according to published procedures such as those described in Parameswara et al., Synthesis, 815-818 (1980) and Denny et al., J. Org. Chem., 27, 3404 (1962).
Carboxylic acid-containing compounds described above can then be converted to hydroxamic acid-containing compounds according to the following scheme: 
Triethylamine (TEA) is added to a cooled (e.g., 0-5xc2x0 C.) anhydrous THF solution containing the carboxylic acid. Isobutyl chloroformate is then added to the solution having carboxylic acid, which is followed by the addition of hydroxylamine hydrochloride and TEA. After acidification, the solution was filtered to collect the desired hydroxamic acid-containing compounds.
An N-substituted hydroxamic acid can be prepared in a similar manner as described above. A corresponding carboxylic acid Axe2x80x94Lxe2x80x2xe2x80x94C(xe2x95x90O)xe2x80x94OH can be converted to an acid chloride by reacting with oxalyl chloride (in appropriate solvents such as methylene chloride and dimethylformamide), which in turn, can be converted to a desired N-substituted hydroxamic acid by reacting the acid chloride with an N-substituted hydroxylamine hydrochloride (e.g., CH3NHOH.HCl) in an alkaline medium (e.g., 40% NaOH (aq)) at a low temperature (e.g., 0-5xc2x0 C.). The desired N-substituted hydroxamic acid can be collected after acidifying the reaction mixture after the reaction has completed (e.g., in 2-3 hours).
As to compounds of the invention wherein X1 is S, they can be prepared according to procedures described in Sandler, S. R. and Karo, W., Organic Functional Group Preparations, Volume III (Academic Press, 1972) at pages 436-437. For preparation of compounds of the invention wherein X2 is xe2x80x94N(Rc)OHxe2x80x94 and X1 is S, see procedures described in U.S. Pat. Nos. 5,112,846; 5,075,330 and 4,981,865.
Compounds of the invention containing an xcex1-keto acid moiety (e.g., when X1 is oxygen and X2 is xe2x80x94C(xe2x95x90O)OM or Axe2x80x94Lxe2x80x2xe2x80x94C(xe2x95x90O)xe2x80x94C(xe2x95x90O)xe2x80x94OM, where A and Lxe2x80x2have been defined above and M can be hydrogen, lower alkyl or a cation such as K+), these compounds can be prepared by procedures based on that described in Schummer et al., Tetrahedron, 43, 9019 (1991). Briefly, the procedure starts with a corresponding aldehyde-containing compound (e.g., Axe2x80x94Lxe2x80x2xe2x80x94C(xe2x95x90O)xe2x80x94H), which is allowed to react with a pyruvic acid in a basic condition (KOH/methanol) at a low temperature (e.g., 0-5xc2x0 C.). Desired products (in the form of a potassium salt) are formed upon warming of the reaction mixture to room temperature.
The compounds described above, as well as their (thio)hydroxamic acid or xcex1-keto acid counterparts, can possess histone deacetylase inhibitory properties.
Note that appropriate protecting groups may be needed to avoid forming side products during the preparation of a compound of the invention. For example, if the linker Lxe2x80x2 contains an amino substituent, it can be first protected by a suitable amino protecting group such as trifluoroacetyl or tert-butoxycarbonyl prior to being treated with reagents such as butyllithium. See, e.g., T. W. Greene, supra, for other suitable protecting groups.
A compound produced by the methods shown above can be purified by flash column chromatography, preparative high performance liquid chromatography, or crystallization.
A pharmaceutical composition can be used to inhibit histone deacetylase in cells and can be used to treat disorders associated with abnormal histone deacetylase activity. Some examples of these disorders are cancers (e.g., leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, cervical cancer, renal cancer, prostate cancer, and breast cancer), hematological disorders (e.g., hemoglobinopathies, thalassemia, and sickle cell anemia) and genetic related metabolic disorders (e.g., cystic fibrosis, peroxisome biogenesis disorder, alpha-l anti-trypsin, and adrenoleukodystrophy). The compounds of this invention can also stimulate hematopoietic cells ex vivo, ameliorating protozoal parasitic infection, accelerate wound healing, and protecting hair follicles.
An effective amount is defined as the amount which is required to confer a therapeutic effect on the treated patient, and is typically determined based on age, surface area, weight, and condition of the patient. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep. 50, 219 (1966). Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, 537 (1970). An effective amount of a compound described herein can range from about 1 mg/kg to about 300 mg/kg. Effective doses will also vary, as recognized by those skilled in the art, dependant on route of administration, excipient usage, and the possibility of co-usage, pre-treatment, or post-treatment, with other therapeutic treatments including use of other chemotherapeutic agents and radiation therapy. Other chemotherapeutic agents that can be co-administered (either simultaneously or sequentially) include, but not limited to, paclitaxel and its derivatives (e.g., taxotere), doxorubicin, L-asparaginase, dacarbazine, amascrine, procarbazine, hexamethylmelamine, mitoxantrone, and gemicitabine.
The pharmaceutical composition may be administered via the parenteral route, including orally, topically, subcutaneously, intraperitoneally, intramuscularly, and intravenously. Examples of parenteral dosage forms include aqueous solutions of the active agent, in a isotonic saline, 5% glucose or other well-known pharmaceutically acceptable excipient. Solubilizing agents such as cyclodextrins, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic compounds. Because some of the compounds described herein can have limited water solubility, a solubilizing agent can be included in the composition to improve the solubility of the compound. For example, the compounds can be solubilized in polyethoxylated castor oil (Cremophor EL(copyright)) and may further contain other solvents, e.g., ethanol. Furthermore, compounds described herein can also be entrapped in liposomes that may contain tumor-directing agents (e.g., monoclonal antibodies having affinity towards tumor cells).
A compound described herein can be formulated into dosage forms for other routes of administration utilizing conventional methods. For example, it can be formulated in a capsule, a gel seal, or a tablet for oral administration. Capsules may contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets may be formulated in accordance with conventional procedures by compressing mixtures of a compound described herein with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. Compounds of this invention can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tableting agent.
The activities of a compound described herein can be evaluated by methods known in the art, e.g., MTT (3-[4,5-dimehtythiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, clonogenic assay, ATP assay, or Extreme Drug Resistance (EDR) assay. See Freuhauf, J. P. and Manetta, A., Chemosensitivity Testing in Gynecologic Malignancies and Breast Cancer 19, 39-52 (1994). The EDR assay, in particular, is useful for evaluating the antitumor and antiproliferative activity of a compound of this invention (see Example 28 below). Cells are treated for four days with compound of the invention. Both untreated and treated cells are pulsed with tritiated thymidine for 24 hours. Radioactivity of each type of cells is then measured and compared. The results are then plotted to generate drug response curves, which allow IC50 values (the concentration of a compound required to inhibit 50% of the population of the treated cells) to be determined.
The histone acetylation activity of a compound described herein can be evaluated in an assay using mouse erythroleukemia cells. Studies are performed with the DS19 mouse erythroleukemia cells maintained in RPMI 1640 medium with 25 mM HEPES buffer and 5% fetal calf serum. The cells are incubated at 37xc2x0 C.
Histones are isolated from cells after incubation for periods of 2 and 24 hours. The cells are centrifuged for 5 minutes at 2000 rpm in the Sorvall SS34 rotor and washed once with phosphate buffered saline. The pellets are suspended in 10 ml lysis buffer (10 mM Tris, 50 mM sodium bisulfite, 1% Triton X-100, 10 mM magnesium chloride, 8.6% sucrose, pH 6.5) and homogenized with six strokes of a Teflon pestle. The solution is centrifuged and the pellet washed once with 5 ml of the lysis buffer and once with 5 ml 10 mM Tris, 13 mM EDTA, pH 7.4. The pellets are extracted with 2xc3x971 mL 0.25N HCl. Histones are precipitated from the combined extracts by the addition of 20 mL acetone and refrigeration overnight. The histones are pelleted by centrifuging at 5000 rpm for 20 minutes in the Sorvall SS34 rotor. The pellets are washed once with 5 mL acetone and protein concentration are quantitated by the Bradford procedure.
Separation of acetylated histones is usually performed with an acetic acid-urea polyacrylamide gel electrophoresis procedure. Resolution of acetylated H4 histones is achieved with 6,25N urea and no detergent as originally described by Panyim and Chalkley, Arch. Biochem. Biophys. 130, 337-346 (1969). 25 xcexcg total histones are applied to a slab gel which is run at 20 ma. The run is continued for a further two hours after the Pyronon Y tracking dye has run off the gel. The gel is stained with Coomassie Blue R. The most rapidly migrating protein band is the unacetylated H4 histone followed by bands with 1, 2, 3 and 4 acetyl groups which can be quantitated by densitometry. The procedure for densitometry involves digital recording using the Alpha Imager 2000, enlargement of the image using the PHOTOSHOP program (Adobe Corp.) on a MACINTOSH computer (Apple Corp.), creation of a hard copy using a laser printer and densitometry by reflectance using the Shimadzu CS9000U densitometer. The percentage of H4 histone in the various acetylated states is expressed as a percentage of the total H4 histone.
The concentration of a compound of the invention required to decrease the unacetylated H4 histone by 50% (i.e., EC50) can then be determined from data obtained using different concentrations of test compounds.
Histone deacetylase inhibitory activity can be measured based on procedures described by Hoffmann et al., Nucleic Acids Res., 27, 2057-2058 (1999). See Example 30 below. Briefly, the assay starts with incubating the isolated histone deacetylase enzyme with a compound of the invention, followed by the addition of a fluorescent-labeled lysine substrate (contains an amino group at the side chain which is available for acetylation). HPLC is used to monitor the labeled substrate. The range of activity of each test compound is preliminarily determined using results obtained from HPLC analyses. IC50 values can then be determined from HPLC results using different concentrations of compounds of this invention. All assays are duplicated or triplicated for accuracy. The histone deacetylase inhibitory activity can be compared with the increased activity of acetylated histone for confirmation.
Compounds of this invention are also evaluated for effects on treating X-linked adrenoleukodystrophy (X-ALD), a peroxisomal disorder with impaired very long-chain fatty acid (VLCFA) metabolism. In such an assay, cell lines derived from human primary fibroblasts and (EBV-transformed lymphocytes) derived from X-ALD patients grown on RPMI are employed. Tissue culture cells are grown in the presence or absence of test compounds. For VLCFA measurements, total lipids are extracted, converted to methyl esters, purified by TLC and subjected to capillary GC analysis as described in Moser et al., Technique in Diagnostic Biochemical Genetics: A Laboratory Manual (ed A., H. F) 177-191 (Wiley-Liss, New York, 1991). C24:0 xcex2-oxidation activity of lyophoclastoid cells are determined by measuring their capacity to degrade [1-14C]-C24:0 fatty acid to water-soluble products as described in Watkins et al., Arch. Biochem. Biophys. 289, 329-336 (1991). The statistical significance of measured biochemical differences between untreated and treated X-ALD cells can be determined by a two-tailed Student""s t-test. See Example 31 below.
Further, compounds of the present invention are evaluated for their effects in treating cystic fibrosis (CF). Since the initial defect in the majority of cases of CF is the inability of mutant CF protein (CFTR) to fold properly and exit the ER, compounds of the invention are tested to evaluate their efficacy in increasing the trafficking of the CF protein out of the ER and its maturation through the Golgi. During its biosynthesis, CFTR is initially synthesized as a nascent polypeptide chain in the rough ER, with a molecular weight of around 120 kDa (Band A). It rapidly receives a core glycosylation in the ER, giving it a molecular weight of around 140 kDa (Band B). As CFTR exits the ER and matures through the Golgi stacks, its glycosylation is modified until it achieves a terminal mature glycosylation, affording it a molecular weight of around 170 kDa (Band C). Thus, the extent to which CFTR exits the ER and traverses the Golgi to reach the plasma membrane may be reflected in the ratio of Band B to Band C protein. CFTR is immunoprecipitated from control cells, and cells exposed to test compounds. Both wt CFTR and xcex94F508 CFTR expressing cells are tested. Following lysis, CFTR are immunoprecipitated using various CFTR antibodies. Immunoprecipitates are then subjected to in vitro phosphorylation using radioactive ATP and exogenous protein kinase A. Samples are subsequently solubilized and resolved by SDS-PAGE. Gels are then dried and subject to autoradiography and phosphor image analysis for quantitation of Bands B and C are determined on a BioRad personal fix image station. See Example 32 below. Furthermore, compounds of this invention can be used to treat homozygous xcex2 thalassemia, a disease in which there is inadequate production of xcex2 globin leading to severe anemia. See Collins et al., Blood, 85(1), 43-49 (1995).
Still further, compounds of the present invention are evaluated for their use as antiprotozoal or antiparasitic agents. The evaluation can be conducted using parasite cultures (e.g., Asexual P. falciparum). See Trager, W. and Jensen, J. B., Science 193, 673-675 (1976). Test compounds of the invention are dissolved in dimethyl sulfoxide (DMSO) and added to wells of a flat-bottomed 96-well microtitre plate containing human serum. Parasite cultures are then added to the wells, whereas control wells only contain parasite cultures. After at least one invasion cycle, and addition of labeled hypoxanthine monohydrochloride, the level of incorporation of labeled hypoxanthine is detected. IC50 values can be calculated from data using a non-linear regression analysis.
The toxicity of a compound described herein is evaluated when a compound of the invention is administered by single intraperitoneal dose to test mice. See Example 33 below. After administration of a predetermined dose to three groups of test mice and untreated controls, mortality/morbidity checks are made daily. Body weight and gross necropsy findings are also monitored. For reference, see Gad, S. C. (ed.), Safety Assessment for Pharmaceuticals (Van Nostrand Reinhold, New York, 1995).
Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific examples, which described syntheses, screening, and biological testing of various compounds of this invention, are therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications recited herein, including patents, are hereby incorporated by reference in their entirety.